The majority of mitochondria are furnished by proteins encoded on nuclear genes. Many of these proteins interact to form multienzyme complexes on the matrix side of the inner membrane. This proposal is designed to elucidate specific steps in gene expression for one of these mitochondrial complexes, human branched chain alpha-ketoacid dehydrogenase [BCKD]. This complex is of interest since inherited mutations occur in humans which decrease the function of BCKD resulting in a phenotype known as maple syrup urine disease [MSUD]. Three gene products are unique for the catalytic components of BCKD and provide the focus of these studies. Five specific aims are addressed related to these three genes. 1. Using cloned DNA fragments for the immediate 600 bp upstream from each of the transcriptional start site of each gene, the cis elements and trans-acting DNA binding proteins will be identified using footprinting and gel retardation analysis. Similarities among the three promoters will be sought. 2. Media conditions for cultured human cells are known which alter the amount of mRNA for the individual subunits. Studies are designed to differentiate whether altered transcription or mRNA stability cause these changes and how the changes affect BCKD activity. 3. Since the proteins assemble into the complex with known stoichiometry, studies to address whether the import of these preproteins affect each other and play a role in developing the stoichiometry. 4. Fetal expression of BCKD has never been studied. Murine embryos will be used to define the pattern of gene expression throughout fetal development. It is important to understand this pattern to provide improved management of pregnancies of the heterozygote and newly emerging homozygous MSUD mothers. 5. Characterization of additional mutations in these genes will lead to a better understanding of genotype/phenotype relations and patient management. Understanding the mutations will direct future studies on the assembly and function of BCKD. An ultimate goal is to provide gene therapy for MSUD.